Fecal lactoferrin as a biomarker for determining disease severity and for treating infection in patients with clostridium difficile disease

ABSTRACT

Clostridium difficile  disease involves a range of clinical presentations ranging from mild to self-limiting diarrhea to life-threatening pseudomembranous colitis and megacolon. Cases of  C. difficile  are treated differently depending on severity of disease. Mild and moderate cases may be treated with metronidazole while moderate-to-severe and relapsing cases are often treated with vancomycin or fidaxomicin. The presence of  C. difficile  disease is detected using a biomarker panel that includes  C. difficile  antigen (GDH), toxins A and B, and fecal lactoferrin. In patients suspected of  C. difficile  disease, if GDH is detected indicating the presence of  C. difficile , and then toxins A and/or B are detected to indicate toxigenic  C. difficile  and support a diagnosis of  C. difficile -associated disease, fecal lactoferrin concentrations are measured to determine severity of the disease by indicating the amount of intestinal inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/480,616, filed Apr. 29, 2011, entitled “Fecal Lactoferrin as aBiomarker for Determining Disease Severity and for Monitoring Infectionin Patients with Clostridium Difficile Disease,” which is hereinincorporated by reference.

BACKGROUND

Clostridium difficile infection (CDI) involves a range of clinicalpresentations including mild to self-limiting diarrhea tolife-threatening pseudomembranous colitis and megacolon. Many healthypersons (e.g., infants) carry Clostridium difficile (C. difficile), andmany patients become asymptomatic carriers after admission to thehospital. Most cases are diagnosed based on clinical evaluations,history of antibiotic use, and the presence of the organism and/ortoxins A & B (i.e., TcdA and TcdB, respectively) in the stool.Enzyme-linked immunoassay (EIA) tests are the most frequently used testformat for measuring toxin in the stool specimens, with tissue culturecombined with specific neutralization being the gold standard fordetecting stool toxin. More recently, polymerase chain reaction (PCR)tests are available for determining the presence of C. difficile toxin Aand B genes (tcdA and tcdB) and these are used as standalone tests andin combination with the detection of glutamate dehydrogenase (GDH) forruling out C. difficile-negative patients. All of these assays aresuitable for detecting the presence of C. difficile as an aid todiagnosis but do not provide information about the severity of disease.

The severity of the disease is an important factor to recommending aproper course of treatment. In general, patients with C. difficiledisease often present with fever, have slightly raised white blood cells(leukocytosis) and experience mild abdominal pain. Mild cases respondwell to stopping the inciting antibiotic while moderate and/ormoderate-to-severe C. difficile disease cases often require antibioticintervention. Currently, no single lab parameter is routinely used tostratify patients based on severity of CDAD for optimizing medicaland/or surgical treatment.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

Illustrative embodiments of the invention are described in detail belowwith reference to the attached drawing figures, wherein:

FIG. 1A depicts patient characteristics for patients diagnosed with C.difficile disease according to embodiments of the invention;

FIG. 1B depicts mean lactoferrin levels (μg/mL±standard error) forpatients with clinically defined cases of C. difficile diseasestratified by severity according to embodiments of the invention;

FIG. 2 depicts mean lactoferrin levels (μg/mL±standard error) forpatients stratified by ARL 027 versus other ribotype C. difficileinfections according to embodiments of the invention;

FIG. 3 depicts daily monitoring of lactoferrin levels during and afterantibiotic treatment in a patient with C. difficile disease according toembodiments of the invention;

FIG. 4A depicts a summary of biomarker results for patients with aclinical cure (no symptoms and no C. difficile during and/or afterinitial treatment) according to embodiments of the invention;

FIG. 4B depicts a summary of biomarker results for patients withbacterial reinfection (return of C. difficile in absence of symptomsduring and /or after initial treatment) according to embodiments of theinvention; and

FIG. 4C depicts a summary of biomarker results for patients withclinical recurrence or no cure (return of symptoms and C. difficileduring and /or after initial treatment) according to embodiments of theinvention.

DETAILED DESCRIPTION

The present invention is directed to test methods for aiding instratifying patients based on severity of C. difficile disease.Stratifying patients with disease based on severity using a panel ofbiomarkers is a new concept that is critically needed because of theincrease in incidence and frequent severe presentations and overuse ofantibiotics. The emergence of the outbreak strain ribotype ARL 027 thatproduces more toxin and spores has been linked with more severe C.difficile disease and a greater chance of relapse. In addition, newermedications like the antibiotic fidaxomicin (Dificid) offer additionaltreatment options for C. difficile disease. In a study published by L.Kyne et al. 1999, the authors performed a detailed characterization ofdisease states for an outbreak of CDAD in Dublin, Ireland. Thisparticular outbreak involved 14 patients that were stool cytotoxinpositive but asymptomatic. Of the symptomatic patients, 25% hadmild-self-limiting disease with no antibiotic treatment, 35% hadmoderately severe C. difficile disease responding to antibiotictreatment and 40% developed severe disease with prolonged symptomslasting between eleven to thirty-six days. A total of 8% of the patientswith C. difficile disease progressed to severe colitis withpseudomembranes and toxic megacolon. The authors noted that physiciansshould be aware of early indicators of disease severity in order tolower morbidity and mortality for cases of C. difficile disease.

A combination of clinical presentations and various lab parameters havebeen evaluated for stratifying patients by disease activity (e.g., mild,moderate, and moderate-to-severe). White blood cell count (WBC), serumalbumin level (indicator of leakage into the bowel), and creatininelevel for monitoring kidney failure are the most commonly used labindicators for disease activity for C. difficile. Mild to moderate casesof C. difficile usually present with a WBC≦15,000/μL, normal serumcreatinine (<2.0 mg/dL) and albumin levels (≧2.5 g/dL). Symptoms includehaving less than 10 watery stools without blood per day and mildcramping lasting for up to an average of 4 days. A common treatment forpatients with an initial episode of mild to moderate C. difficiledisease is treatment with a member of the nitroimidazole class ofantibiotics. For example, mild to moderate C. difficile disease may betreated with 500 mg metronidazole, three times daily for ten days. Mostcases resolve with no further complications, but up to 25% of thesecases may relapse multiple times and require a second round ofantibiotics, which historically has included treatment with a member ofthe glycopeptide class of antibiotics, such as vancomycin. However, nowsuch second rounds of antibiotics include members of the macrocyclicclass of antibiotics, such as fidaxomicin (Dificid).

Patients over the age of sixty-five with multiple co-morbidities are ata higher risk for C. difficile disease and more often suffer from moresevere disease leading to multiple relapses. Severe fulminant C.difficile disease is characterized by having eleven or more liquidstools per day for more than ten days. Stool specimens often containmucus and may be bloody. Defined lab parameters for fulminant C.difficile colitis are WBC≧15,000/μL, a rising serum creatinine (50%increase and levels≧2.0 mg/dL) indicating poor kidney function andalbumin levels dropping below 2.5 g/dL showing loss of protein becauseof exudation of serum into the bowel. Clinical presentations may involvepseudomembranes on endoscopy, severe abdominal pain and cramping, andcolonic thickening observed by CT scan. Toxic megacolon stemming fromileus may occur causing nausea, vomiting, severe dehydration, andextreme lethargy. Treatment for severe and relapsing cases of C.difficile disease usually involves 125 mg vancomycin 4 times per day for10 days.

Identifying disease activity for patients with C. difficile infection isimperative for proper treatment and better outcome with decreasedmorbidity and mortality. An embodiment of the invention provides adiagnostic parameter for assessing severity in C. difficile disease bymeasuring fecal lactoferrin and using the measurement of fecallactoferrin as an indicator for intestinal inflammation caused by C.difficile.

C. difficile disease is an inflammatory disease involving theinfiltration of activated neutrophils across the mucosa into the lumencausing colitis and in severe cases, the formation of pseudomembranes.Human lactoferrin is a glycoprotein that is present in most mucosalsecretions and a primary component of the granules of activatedneutrophils. During the onset of intestinal inflammation from C.difficile, activated neutrophils infiltrate the intestinal lumen causingan increase in fecal lactoferrin.

Fecal specimens are routinely collected for C. difficile testing(antigen and toxin). Accordingly, additional testing can be done tomeasure the level of fecal lactoferrin for determining the amount ofintestinal inflammation as an indicator of disease severity. Inaddition, combining the presence of antigen and the levels of toxins Aand B with fecal lactoferrin concentrations can help the physician indetermining if a patient is a carrier from patients that have true mildto severe infections for optimal medical treatment.

In an embodiment of the present invention, a method for assessingdisease severity in patients with C. difficile disease using fecallactoferrin levels is provided. Toxin A is a strong chemotactic proteinthat causes the release of IL-8 and the infiltration of activatedneutrophils into the intestinal mucosa. In fact, toxin A concentrationsof 100-fold less than that of toxin B have been shown to stimulate therelease of IL-8. Toxin A also stimulates other pro-inflammatorycytokines including I1-1β and tumor necrosis factor alpha (TNF-{acuteover (α)}). Toxin B is a cytotoxin that causes tissue damage andinflammation that contributes, along with toxin A that causes fluidaccumulation, to disease. The combined effects of the enterotoxic andchemotactic toxin A and cytotoxic effects of toxin B strongly contributeto the severity of disease. In a study by Kuehne et al., knockoutmutants showed that both A+B− and A−B+ mutants were cytotoxic and causeddisease in the hamster model. An interesting finding was that when tcdBwas inactivated by an insertion, the resulting A+B− mutant showedincreased cytotoxicity of toxin A in cell culture. The increasedcytotoxicity was not neutralized completely by anti-toxin A specificantibody. The reason for the increase of cytotoxicity following theinactivation of tcdB was not determined but thought to be due toincreased expression. The double knockout mutant A−B− did not causedisease in the hamster. These results confirmed that both TcdA and TcdBin combination and independently cause disease. In another study, theanalysis of A−B+ isolates showed a variant toxin B that wassignificantly more lethal in a mouse than normal toxin B. These studiessupport the role of both toxins in the disease. A method for determiningthe presence of intestinal inflammation in combination with the presencetoxin in stool can offer additional information on disease status forpatients with C. difficile infection.

An embodiment of the present invention provides for determining thepresence of C. difficile disease using a biomarker panel that includes,by way of example, C. difficile antigen (GDH), toxins A (tcdA or TcdA)and B (tcdB or TcdB) for determining the presence of toxigenic C.difficile. As will be understood, further embodiments of the inventionutilize additional biomarkers for C. difficile infection. When adiagnosis of C. difficile disease is concluded, fecal lactoferrinconcentrations may be used to determine disease severity. In patientssuspected of infection with C. difficile, if GDH is present, indicatingthe presence of C. difficile, then toxins A and/or B (genes and/orprotein) are detected to show the presence of toxigenic C. difficilefollowed by measuring fecal lactoferrin levels as an indicator ofintestinal inflammation. Knowing whether toxigenic C. difficile ispresent in combination with a lactoferrin concentration will help todetermine disease severity to optimize treatment.

In embodiments, serial measurements of biomarkers for C. difficileinfection are utilized. For example, lactoferrin, GDH, toxin A, and/ortoxin B levels may be monitored at regular intervals during analysisand/or treatment. In embodiments, serial analysis of the presence of oneor more biomarkers (e.g. GDH, toxins A and/or B) provides an indicatorof the bacteria, which may be used to determine a patient's response totreatment.

In embodiments, the level of lactoferrin in fecal samples provides anindication of the severity of C. difficile. For example, “mild” C.difficile disease may be indicated in samples with less than 7.25 μg/mLlactoferrin. In embodiments, a diagnosis of mild C. difficile disease isindicated in samples with less than 7.25 μg/mL lactoferrin, combinedwith clinical indicators for defining the mild disease. For example,clinical indicators such as the number of unformed stools per day, apresence of fever, abdominal pain, and vomiting may be characterizedand/or determined as being indicative of a diagnosis of mild C.difficile disease, and may be analyzed together with a measurement ofless than 7.25 μg/mL lactoferrin, to determine disease severity. Inembodiments, clinical indicators for a diagnosis of mild C. difficileinclude having three to five stools per day and a white blood cell countless than or equal to 15,000/mm³. In further embodiments, lab parameterssuch as C-reactive protein (CRP), white blood cell count (WBC), serumalbumin, and/or creatinine, may be combined with a level of lactoferrin,a level of calprotectin, and/or a clinical indicator(s) to determinedisease severity in patients diagnosed with mild C. difficile.

In another example, “moderate” C. difficile disease may be indicated insamples with between 7.25 μg/mL to 99.99 μg/mL lactoferrin. In someembodiments, a diagnosis of moderate C. difficile disease is indicatedin samples with between 7.25 μg/mL to 99.99 μg/mL lactoferrin, combinedwith clinical indicators for defining the moderate disease. For example,clinical indicators such as the number of unformed stools per day, apresence of fever, abdominal pain, and vomiting may be characterizedand/or determined as being indicative of a diagnosis of moderate C.difficile disease, and may be analyzed together with a measurementbetween 7.25 μg/mL to 99.99 μg/mL lactoferrin, to determine diseaseseverity. In embodiments, clinical indicators for a diagnosis ofmoderate C. difficile include having six to nine stools per day, a whiteblood cell count from 15,001/mm³ to 20,000/mm³, and moderate abdominalpain. In further embodiments, lab parameters such as C-reactive protein(CRP), white blood cell count (WBC), serum albumin, and/or creatinine,may be combined with a level of lactoferrin, a level of calprotectin,and/or a clinical indicator(s) to determine disease severity in patientsdiagnosed with moderate C. difficile.

In a further example, “moderate-to-severe” C. difficile disease may beindicated in samples with 100 μg/mL or greater lactoferrin. In someembodiments, a diagnosis of moderate-to-severe C. difficile disease isindicated in samples with 100 μg/mL or greater lactoferrin, combinedwith clinical indicators for defining the moderate-to-severe disease.For example, clinical indicators such as the number of unformed stoolsper day, a presence of fever, abdominal pain, and vomiting may becharacterized and/or determined as being indicative of a diagnosis ofmoderate-to-severe C. difficile disease, and may be analyzed togetherwith a measurement of 100 μg/mL or greater lactoferrin, to determinedisease severity. In embodiments, clinical indicators for a diagnosis ofmoderate-to-severe C. difficile include having ten or greater stools perday, a white blood cell count of 20,001/mm³ or greater, and severeabdominal pain. In further embodiments, lab parameters such asC-reactive protein (CRP), white blood cell count (WBC), serum albumin,and/or creatinine, may be combined with a level of lactoferrin, a levelof calprotectin, and/or a clinical indicator(s) to determine diseaseseverity in patients diagnosed with moderate-to-severe C. difficile.

One exemplary method of testing for the presence of the C. difficile GDHbiomarker is to use the C. DIFF CHEK™-60 test, which uses antibodiesspecific for C. difficile GDH. The Microassay Plate contains immobilizedpolyclonal antibody against the GDH antigen, while the Conjugateconsists of a highly specific monoclonal antibody conjugated tohorseradish peroxide. If the GDH antigen is present in the specimen, acolor is detected due to the enzyme-antibody-antigen complexes that formin the assay.

One exemplary method of testing for the presence of toxin A and toxin Bis to use the C. DIFFICILE TOX A/B II™ test, which uses antibodies to C.difficile toxins A and B. The test utilizes immobilizedaffinity-purified polyclonal antibody against toxins A and B, and thedetecting antibody consists of a mixture of toxin A monoclonal antibodyconjugated to horseradish peroxidase and toxin B polyclonal antibodyconjugated to horseradish peroxidase. If toxins A and B are present inthe specimen, a color is detected due to the enzyme-antibody-antigencomplexes that form in the assay.

One exemplary method of testing the presence of GDH, toxin A and toxin Bis to use the QUIK CHEK COMPLETE™ test, which uses antibodies specificfor GDH and toxins A and B of C. difficile. The device contains threevertical lines of immobilized antibodies, the antigen test line containsantibodies against C. difficile GDH, and the control line is a dottedline that contains anti-horseradish peroxidase antibodies. The toxins Aand B test line contains antibodies against C. difficile toxins A and Band the Conjugate consists of antibodies to GDH and antibodies to toxinsA and B coupled to horseradish peroxidase. The GDH reaction is examinedvisually for the appearance of a vertical blue line, which indicates apositive test, while a blue line also indicates a positive test fortoxin A and toxin B.

One exemplary method of testing for the presence of C. difficile toxinis the C. DIFFICILE TOX-B TEST™, which uses a tissue culture format todetect the presence of cytotoxic activity in fecal specimens andconfirms the identification of C. difficile toxin using specificantitoxin. The test confirms the presence of C. difficile toxin byneutralizing the cytotoxic activity with a reagent that is a specificantitoxin. In the assay, if C. difficile toxin is present, the cells inthe well with PBS will become round, demonstrating the presence of thecytotoxic activity, while the presence of C. difficile toxin isconfirmed if the cytotoxic activity is neutralized in the wellcontaining antitoxin.

One exemplary method of treating C. difficile is through a native floratransplant. This process, also referred to as Fecal (or Faecal)Microbiota Transplantation (FMT), is the restoration of the colonicflora by introducing healthy bacterial flora through infusion of stool,e.g. by enema, obtained from a healthy human donor. A native floratransplant can also be administered as a liquid that the patient drinks.

The following are examples of procedures which have been utilized toestablish the preferred assays according to the present invention. Thefollowing examples are merely exemplary and not presented by way oflimitation.

EXAMPLE 1

Fecal lactoferrin levels were evaluated in patients with clinicallydefined C. difficile disease ranging from mild to moderate-to-severedisease. Briefly, patients with clinically confirmed C. difficiledisease presenting with a spectrum of severity were recruited along withfourteen age-sex matched healthy subjects defined as having nointestinal illnesses. Disease activity was defined by physician'sassessment and based on symptoms, serum albumin, WBC counts andco-morbidities. Fecal lactoferrin was measured using a quantitativeenzyme immunoassay (EIA). C. difficile glutamate dehydrogenase (GDH) andtoxins A and B in stool were detected using a membrane-based EIA.Toxigenic culture was done using spore enrichment and both isolates andstool specimens were tested by tissue culture assay for cytotoxicity.

RESULTS

Thirty-nine clinically confirmed cases of C. difficile disease (fifteenmoderate-to-severe, twenty-one moderate and three mild) were testedduring a six month period. Ages ranged from thirty-two to eighty-nineyears and fifty percent were female. The predominant co-morbidities werediabetes (31%), cancer (23%) and renal failure (23%). All patients wereGDH-positive and toxigenic C. difficile was isolated from all but fourpatients. The mean lactoferrin levels (μg/mL±std error) were 1198±404for moderate-to-severe, 132±50 for moderate, 12±5 for mild and 2±0.3 forhealthy subjects. Stool toxin was detected by tissue culture in 87% (13/15) of moderate-to-severe, 71% ( 15/21) of moderate and 33% (⅓) formild disease. Two of the moderate-to-severe patients with the lowestlactoferrin levels (≦8 μg/mL) and three of the four lowest with moderate(≦12 μg/mL) were also tissue culture-negative. Of these patients, bothof the severe and two of the four moderate patients had negative stoolcultures. All of these patients had co-morbidities that contributed tothe clinical assessments. Our conclusion is that in a clinical setting,co-morbidities can complicate the clinical assessment for C. difficileinfection. Our results show that fecal lactoferrin is useful forindicating disease severity in patients with C. difficile infection.

Accordingly, FIG. 1A details the patient characteristics for clinicallyconfirmed cases of C. difficile disease. Most patients were >64 yearsold, experienced pain, had liquid stools and suffered withco-morbidities including diabetes, cancer, renal failure and pneumonia.FIG. 1B shows that lactoferrin levels were significantly higher betweenmild, moderate, and moderate-to-severe cases of C. difficile disease,and trended higher for the moderate-to-severe group.

FIG. 2 shows the mean lactoferrin levels for patients with clinicallyconfirmed C. difficile disease grouped by ribotype. Patients infectedwith ARL 027 had significantly higher levels of lactoferrin thanpatients infected with other ribotypes. Studies have shown that patientsinfected with ARL 027 tend to have stool toxin and present with moresevere disease.

EXAMPLE 2

Fecal C. difficile GDH, toxins A and B, and human lactoferrin levelswere measured in several subjects with C. difficile disease duringantibiotic treatment. Both subjects with clinically confirmed C.difficile disease were monitored for the presence of GDH, toxins A and Band fecal lactoferrin by enzyme-linked immunoassay (EIA). Specimencollection was initiated at the start of antibiotic treatment and wascontinued on a daily to weekly basis when possible. A symptom log waskept by each patient and all treatments were recorded during the testperiod. Both patients showed a rapid response to antibiotic treatmentwith fecal GDH, toxins A and B, and fecal lactoferrin reaching baselinewithin 24 hours. Antigen, toxin and fecal lactoferrin remained negativeduring the antibiotic therapy. Following the treatment, both patientsexperienced a clinical relapse and showed a rapid increase for allparameters. Following a second course of antibiotics, all parametersreturned to baseline. At completion of the second course of antibiotics,all parameters increased rapidly in absence of clinical symptoms. BothGDH and toxin remained present for 3 to 4 weeks but fecal lactoferrinquickly returned to baseline. No antibiotics were administered sincethere were no clinical symptoms and patients remained healthy.

RESULTS

In this evaluation, it was observed that C. difficile GDH, toxin andfecal lactoferrin levels responded quickly to antibiotic therapy byreturning to baseline (negative). More interestingly, both GDH and toxinwere present without clinical symptoms and with no intestinalinflammation as determined by baseline lactoferrin. These results show arole for fecal lactoferrin in combination with antigen and toxinmeasurements for determining which cases of C. difficile disease mayrequire no further treatment with antibiotics. In addition, ourinvention provides a role for fecal lactoferrin in monitoring C.difficile disease. By determining the amount of intestinal inflammationusing lactoferrin in C. difficile disease patients along with clinicalassessments, the identification of patients for severity of disease mayprove useful for optimizing treatment and leading to better patientoutcomes.

Treatment may be optimized for C. difficile disease since varying levelsof severity call for different treatment recommendations. For example,mild cases of C. difficile disease often receive no antibiotictreatment. In contrast, a case of moderate severity may call for anantibiotic such as metronidazole while a moderate-to-severe case of C.difficile disease may be treated with antibiotics such as vancomycin andfidaxomicin (Dificid).

FIG. 3 illustrates daily lactoferrin levels from the initial episode ofC. difficile infection, during, and after antibiotic treatment.Lactoferrin was elevated (≧7.25 μg/mL) during the initial episode andfor both periods of relapse. Levels drop rapidly once treatment isstarted and increased as symptoms return.

EXAMPLE 3

Patients (pts) with diarrhea and positive stool toxin (TcdA and TcdB)and/or glutamate dehydrogenase (GDH) were recruited with InformedConsent. Stool specimens were collected starting at admission (T=0) toFollow-up (T=F). GDH, toxin, and lactoferrin (LF: median μg/g) weremeasured in stool specimens by immunoassay. Bacterial culture and counts(median CFU#/g) were done using ethanol enrichment and isolates wereribotyped. A total of 18 inpatients were recruited and followed for amedian period of 21 days from T=0 to T=F. Median age was 75 yr and themale:female ratio was 1:3.5. Pts were stratified into 3 groups (i) ptswho were treated and showed no recurrence (N=9). (ii) pts who weretreated with complete resolution of symptoms but had CDI (N=5) and (iii)pts that responded initially to treatment but relapsed (N=4).

RESULTS

Patients in group (i) went from strongly positive for GDH, toxin and aspore count of 10⁴ at T=0 to negative for all biomarkers at T=F. LF fellfrom 406 to 4 during this period (Table 1a). Four of the 5 pts in group(ii) were positive for GDH, toxin, and had a spore count of 10⁴ at T=0.At T=F, 3 of the 5 pts were toxin negative, 3 pts remained GDH-positiveand all pts had spores (10³). LF for these pts dropped from 85 to 2associated with resolution of symptoms (Table 1b). For group (iii), all4 pts remained symptomatic and stayed strongly positive for GDH, toxin,and had a spore count of 10⁴. LF levels for this group were similar atboth T=0 and T=F (362 and 315, respectively) (Table 1c). A total of 5(28%) pts had 027 CDI at T=0. In group (ii), 3 of 5 pts were reinfectedwith 027 as carriers. In group (iii), 1 patient converted to 027. **Allof the 027 isolates were fluoroquinolone resistant. In our study, at T=F50% of pts had no CDI, 28% became carriers and 22% remained ill. GDH,toxin and LF levels all correlated with the presence of clinicaldisease. C. difficile continues to be a complex infection, and accuratediagnosis of disease relies on the clinical history used in conjunctionwith biomarkers for the organism and for inflammation.

FIG. 4A shows the results of CDI biomarkers before and after antibiotictreatment for C. difficile disease. All of the patients in this grouphad a clinical cure meaning no symptoms and no C. difficile detectedduring and after initial antibiotic treatment.

FIG. 4B shows the results of CDI biomarkers before and after antibiotictreatment for C. difficile disease. All patients in this group had areinfection of C. difficile meaning that the C. difficile organism wasdetected in absence of symptoms during and/or after initial antibiotictreatment.

FIG. 4C shows the results of CDI biomarkers before and after antibiotictreatment for C. difficile disease. All patients in this group had aclinical recurrence or no cure meaning that symptoms and the C.difficile organism was maintained or returned during and/or afterinitial antibiotic treatment.

In an alternative embodiment, fecal calprotectin may be utilized ratherthan, or in addition to, fecal lactoferrin as a non-invasive marker formeasuring intestinal inflammation. For example, in a person diagnosedwith C. difficile disease, a quantitative level of fecal calprotectinmay be measured and the quantitative level may be associated with adisease severity including mild, moderate, and moderate-to-severe.Further, fecal calprotectin may be measured subsequent to treatment tomonitor a person's response to medical treatment or an activity level ofthe disease.

In summary, the present invention is directed to non-invasive methodsfor identifying a severity of C. difficile disease in persons diagnosedwith C. difficile disease using lactoferrin. The identified diseaseseverity may be used to recommend a preferred course of treatment forthe person. The present invention is further directed to utilizingchanges in lactoferrin levels to monitor a person's disease activityand/or response to treatment.

The immunoassays of the present invention detect lactoferrin, a stableprotein that serves as an indicator of intestinal inflammation, andprovide quantitative fecal lactoferrin levels for associating a diseaseseverity to C. difficile disease and for monitoring disease activity.The present invention has been described in relation to particularembodiments which are intended in all respects to be illustrative ratherthan restrictive. Alternative embodiments will become apparent to thoseskilled in the art to which the present invention pertains withoutdeparting from its scope.

From the foregoing, it will be seen that this invention is one welladapted to attain all the ends and objects herein above set forthtogether with other advantages which are obvious and which are inherentto the method. It will be understood that certain features andsubcombinations are of utility and may be employed without reference toother features and subcombinations. This is contemplated by and iswithin the scope of the claims.

1. A method of treating a patient with C. Difficile disease, the methodcomprising: administering a therapeutically effective amount oftreatment shown to be effective in treating moderate-to-severe C.Difficile to a patient who has tested positive for an elevated level ofone or more of lactoferrin or calprotectin in a fecal sample indicatingsevere intestinal inflammation.
 2. The method of claim 1, wherein thelevel of one or more of lactoferrin or calprotectin in the fecal sampleis determined through use of a quantitative enzyme immunoassay (EIA). 3.The method of claim 1, wherein the elevated level of lactoferrin is 100μg/mL or greater in the fecal sample.
 4. The method of claim 1, whereinthe therapeutically effective treatment is one or more of glycopeptideantibiotics or macrocyclic antibiotics.
 5. The method of claim 1,wherein the therapeutic treatment is a native flora transplant.
 6. Amethod of treating a patient with C. Difficile disease, the methodcomprising: administering a therapeutically effective amount oftreatment shown to be effective in treating moderate C. Difficile to apatient who has tested positive for an elevated level of one or more oflactoferrin or calprotectin in a fecal sample indicating moderateintestinal inflammation.
 7. The method of claim 6, wherein the level ofone or more of lactoferrin or calprotectin in the fecal sample isdetermined through use of a quantitative enzyme immunoassay (EIA). 8.The method of claim 6, wherein the elevated level of lactoferrin is 7.25μg/mL to 99.99 μg/mL in the fecal sample.
 9. The method of claim 6,wherein the therapeutically effective treatment is one or morenitroimidazole antibiotics.
 10. The method of claim 6, wherein thetherapeutically effective treatment is a native flora transplant.
 11. Amethod of diagnosing a patient with a severity of Clostridium Difficiledisease, the method comprising: obtaining a fecal sample from a personhaving been diagnosed with C. difficile disease; determining whether anelevated level of one or more of lactoferrin or calprotectin is presentin the fecal sample; and upon determining that an elevated level of oneor more of lactoferrin or calprotectin is present in the fecal sample,identifying a severity of the C. difficile disease based on a level ofone or more of lactoferrin or calprotectin present in the fecal sample.12. The method of claim 11, wherein the level of one or more oflactoferrin or calprotectin in the fecal sample is determined throughuse of a quantitative enzyme immunoassay (EIA).
 13. The method of claim11, wherein the elevated levels of lactoferrin is 100 μg/mL or greaterin the fecal sample and the severity of C. Difficile disease isconsidered moderate-to-severe.
 14. The method of claim 13, wherein thetherapeutically effective treatment is one or more of glycopeptideantibiotics or macrocyclic antibiotics.
 15. The method of claim 13,wherein the therapeutically effective treatment is a native floratransplant.
 16. The method of claim 11, wherein the elevated levels oflactoferrin is 7.25 μg/mL to 99.99 μg/mL in the fecal sample and theseverity of C. Difficile disease is considered moderate.
 17. The methodof claim 16, wherein the therapeutically effective treatment is one ormore nitroimidazole antibiotics.
 18. The method of claim 11, wherein theelevated levels of lactoferrin is less than 7.25 μg/mL in the fecalsample and the severity of C. Difficile disease is considered mild,wherein the therapeutically effective treatment is no treatment.
 19. Themethod of claim 11, wherein the level of one or more of lactoferrin orcalprotectin used to determine disease severity in patients diagnosedwith C. difficile is combined with one or more of the following:clinical symptoms comprising one or more of a number of unformed stoolsper day, a presence of fever, abdominal pain, and vomiting, and labparameters comprising one or more of C-reactive protein (CRP), whiteblood cell count (WBC), serum albumin, and creatinine.
 20. The method ofclaim 11, wherein the diagnosis of C. difficile was concluded based uponpresence of a biomarker that indicates a presence of toxigenic C.difficile.